Expansion-Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) is a powerful technique that integrates expansion microscopy with fluorescence in situ hybridization (FISH) to investigate gene expression in thick tissue samples. The method was originally developed by the MultiFISH Project Team in mouse brain sections (Wang, Eddison et al., 2021, doi:10.1016/j.cell.2021.11.024), but it can be applied to various species and tissues. PTR developed an optimized protocol for the Drosophila central nervous system (CNS).
The fly EASI-FISH protocol allows multiple rounds of hybridization and is suitable for high throughput experimentation. Up to six brains or two CNS can be embedded in a single gel for expansion and FISH, and several gels can be processed in parallel. We use a new gel recipe that improves gel robustness. The embedding of multiple brains in a single gel allows for optimal comparison of experimental conditions and reduces reagent cost. Using the GAL4-UAS system for co-detection of green fluorescent protein (GFP), gene expression can be visualized in specific cell types. Due to the high resolution achieved by expansion microscopy and the sensitivity of the method, single transcripts can be detected, and spot counting can be used to quantify expression levels. Thus, this method combines high image quality and high throughput.
Kari Close, Yisheng He, Gudrun Ihrke, Mark Eddison (2025). Multiplex Detection of Gene Expression in the Intact Drosophila Brain using EASI- FISH. dx.doi.org/10.17504/protocols.io.5jyl8jmw7g2w/v2
Kari Close, Yisheng He, Jennifer Jeter, Gudrun Ihrke, Mark Eddison (2025). Multiplex Detection of Gene Expression in the Intact Drosophila Brain Using Expansion-Assisted Iterative Fluorescence In Situ Hybridization. J. Vis. Exp. 219, e67656, DOI: 10.3791/67656